Transcription initiation by RNA polymerase II (RNAP II) requires distinct classes of transcription factors. These include (i) general transcription factors (GTFs) that are required for initiation at all or most promoters; (ii) promoter-specific factors, including both activators and repressors, that affect the rate of transcription; and (iii) cofactors that mediate either activation or repression. The GTFs include TBP (TATA binding protein), TFIIB, TFIIE, TFIIF and TFIIH, and are required for accurate initiation by RNAP II in vitro. The GTFs are also the direct targets of activators and repressors, as well as their cofactors. A remarkable feature of the GTFs is that they are highly conserved in structure and function among eukaryotic organisms. This expands the repertoire of experimental approaches available to study transcriptional mechanisms. The yeast Saccharomyces cerevisiae is an especially valuable tool for such studies because of its ability to be manipulated by extraordinarily powerful genetic methods. Accordingly, yeast genetics can be used to investigate mechanisms controlling transcription, with the outcome relevant for understanding transcriptional control in higher organisms. The objective of this proposal is to characterize the mechanisms that regulate eukaryotic gene expression, focusing on the roles played by the GTFs. A powerful combination of genetic and biochemical methods are proposed to study the processes affecting both the accuracy and rate of transcription by RNA pol II in yeast The proposal will focus on the roles of TFIIB and a novel protein, designated Ssu72, that interacts with TFIIB. Specific Aims are (I) to define the mechanism of transcription start site selection; (2) to define the role of TFIIB in transcriptional activation; (3) to define the role of Ssu72 in both the accuracy and rate of transcription; and (4) to identify additional factors that affect either the rate or accuracy of initiation. The health relatedness of this proposal is readily apparent since gene expression is regulated primarily at the level of transcription and many human diseases, especially many forms of cancer, are known to be a consequence of aberrant gene expression.